The alveolar epithelium constitutes a functional barrier that limits the leakage of solutes and water from the interstitial and vascular compartments of the lung and actively reabsorbs sodium in a vectorial fashion, leading to net fluid clearance from alveolar air spaces. Alveolar epithelial type I (ATI) cells cover the great majority of the internal surface area of the lung. Despite a presumptive role for ATI cells in alveolar epithelial barrier function and transepithelial Na transport, specific Na transporters have not been localized to ATI cells in situ and the specific contributions of ATI cells to mechanisms of alveolar fluid clearance have not been established. In this proposal, we will exploit our recent success in isolating highly purified ATI cells to elucidate the relative roles of ATI and ATII pneumocytes in alveolar epithelial barrier properties. We hypothesize that 1) alveolar epithelial ATI and ATII cells both participate in active transepithelial ion transport (and therefor alveolar fluid clearance) across the alveolar epithelium and 2) their relative contributions to alveolar fluid homeostasis are reflected in part by their expression of different levels and/or types of ion channels and Na pumps. We will investigate these hypotheses by addressing he following Specific Aims: 1) development of improved approaches for isolating and characterizing ATI cells, 2) analysis of ion transport properties (gene expression and function of Na channels, pumps and other transporters) of freshly isolated ATI and ATII cells, 3) characterization of Na transporters in ATI cells in whole lung in situ by patch clamp analysis and newer localization techniques, and 4) development and study of barrier properties of ATI and ATII cell monolayers. Complementary approaches will be used in isolated ATI and ATII cells, in whole lung, and in ATI and ATII cells in monolayer cultures in order to characterize the relative contributions of ATI and ATII cells to alveolar epithelial barrier function. Elucidating the roles of ATI (and ATII) cells is consistent with our long-term objectives of understanding the mechanisms involved in regulation of alveolar homeostasis and may help lead to new therapeutic maneuvers for modulating alveolar fluid clearance.